Amantadine as a regulator of internal ribosome entry site.
OBJECTIVE
Studies of eukaryotes have yielded 2 translation initiation mechanisms: a classical cap-dependent mechanism and a cap-independent mechanism proceeding through the internal ribosomal entry site (IRES). We hypothesized that it might be possible to identify compounds that may distinguish between cap-dependent translation and cap-independent IRES-mediated translation.


METHODS
To facilitate compound screening, we developed bicistronic reporter constructs containing a beta-galactosidase gene (beta-gal) and a secreted human placental alkaline phosphatase (SEAP) reporter gene. Following transcription, the beta-gal gene is translated by a cap-dependent mechanism, while SEAP expression is controlled by the IRES derived from either enterovirus 71 (EV-71) or encephalomyocarditis virus (EMCV). This assay could potentially identify compounds that inhibit SEAP expression (cap-independent) without affecting beta-gal activity (cap-dependent).


RESULTS
Using a bicistronic plasmid-based transient transfection assay in the COS-1 cells, we identified amantadine, a compound that inhibited the IRES of EV71- and EMCV-mediated cap-independent translation but did not interfere with cap-dependent translation when the dose of amantadine was lower than 0.25 mg/mL.


CONCLUSIONS
These results imply that amantadine may distinguish between cap-dependent translation and cap-independent IRES-mediated translation and can be used to regulate gene expression at a translational level.
Chen
Ying-ju
YJ
Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung-Li, Taiwan, China.
Zeng
Shih-jhan
SJ
Hsu
John Ta
JT
Horng
Jim-tong
JT
Yang
Hong-ming
HM
Shih
Shin-ru
SR
Chu
Yu-ting
YT
Wu
Tzong-yuan
TY
D000547:Amantadine / Q000494:pharmacology*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004353:Drug Evaluation, Preclinical; D004686:Encephalomyocarditis virus / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D016898:Interferon-alpha / Q000494:pharmacology; D039502:Nuclear Cap-Binding Protein Complex / Q000187:drug effects*; D012270:Ribosomes / Q000187:drug effects*; D001616:beta-Galactosidase / Q000378:metabolism
D000998:Antiviral Agents; D016898:Interferon-alpha; D039502:Nuclear Cap-Binding Protein Complex; D000547:Amantadine; D001616:beta-Galactosidase